According to the present state of the art about 50 different partially overlapping ca. 80 mer oligonucleotides have to be firstly synthesized and purified for the synthesis of an approximately 2.5 kb nucleic acid sequence. These are then hybridized in pairs or in subsets and filled in by means of a Klenow polymerase reaction or are constructed in a polymerase chain reaction (PCR) using the external oligonucleotides as primers and unidirectionally linked together (usually by means of restriction sites that have to be incorporated). This method is known as the gap filling method. Alternatively gene fragments can be synthesized by enzymatic or chemical ligation; these fragments can then be assembled to form larger gene sections after purification and/or cloning (so-called cassette method). Both procedures require at least one week in an ideal case but usually require closer to 6-12 weeks and even 6 months. Sequential processes bound to solid phases only have low yields due to the many reaction steps that are required and are therefore also very unreliable.
One of the main problems is that longer oligonucleotides always have an unavoidable portion of termination products due to the coupling efficiency which only reaches 99% per step even in syntheses which progress well. Furthermore deletions also occur which result from non-100% capping. Even in very good syntheses this portion is about 0.25% per coupling step. The separation of the trityl protective groups after completion of the synthesis also does not proceed completely. The incomplete oligonucleotide products that are formed in this manner cannot be completely separated from longer oligonucleotides even with much effort.
With an average coupling efficiency of 98%, one for example obtains a yield of the desired product of complete length of only 19.86% in the case of an 80 mer. With the currently available purification methods the desired end-product can at best be obtained in a purity of 95%. Even if only a small portion of the finally purified oligonucleotides is defective, the probability of a defective final sequence increases dramatically with the number of oligonucleotides that are used. Hence a sequence which is composed of 50 of the described oligonucleotides is only correct in 7.7% of all cases and therefore usually has to be re-worked. This does not take into account the relatively rare incorporation of false bases due to false coupling during the synthesis.
Due to the variety of potential sequences of even relatively short oligonucleotides (there are over 1018 possible sequence variants even of a 30 mer) it is also practically impossible to reuse oligonucleotides for various gene constructs. Hence it is technically not feasible to have available all the oligonucleotides required to generate any sequences. New oligonucleotides have to be synthesized and purified for each new gene construct. However, only a fraction of the synthesized material is actually used for the gene synthesis, the remainder cannot be utilized due to the reasons described above. The unsolved incorporation of oligonucleotide synthesis and purification in the process of gene synthesis is one of the main obstacles to a complete automation of this process which at present is technically extremely difficult and probably practically impossible to accomplish.